Confocal scanning microscope

ABSTRACT

A confocal scanning microscope, having a light source ( 1 ) for illuminating an object ( 6 ), which is to be investigated, with exciting light ( 2 ), at least two detection channels exhibiting detection light ( 8, 9 ) being produced, is configured with regard to a high signal yield and a high signal-to-noise ratio in such a way that at least two detection channels can be optically superimposed by means of a superimposing device ( 11, 12, 13, 15, 17, 18 ).

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This invention claims priority of a German patent application DEP 100 04 233.3 filed Feb. 1, 2000 which is incorporated by referenceherein.

FIELD OF THE INVENTION

[0002] The invention relates to a confocal scanning microscope, having alight source for illuminating an object, which is to be investigated,with exciting light, at least two detection channels exhibitingdetection light being produced.

BACKGROUND OF THE INVENTION

[0003] Microscopes of the type mentioned at the beginning are known inpractice and exist in the most varied embodiments. An example of such amicroscope is formed by a confocal scanning microscope in the case ofwhich an object to be investigated is scanned with the aid of a lightbeam or the exciting light. The microscope generally comprises a lightsource and a focussing optical system with the aid of which the lightfrom the source is focussed onto a pinhole stop. In this case, a beamsplitter, a scanning device for beam control, a microscope opticalsystem, a detection stop and detectors for detecting detection and/orfluorescent light are provided.

[0004] The illuminating light is mostly coupled in via the beamsplitter. The focus of the light beam is moved with the aid of thescanning device in a sample plane. For this purpose, it is customary touse two mirrors which are tilted, the deflection axes mostly beingperpendicular to one another, such that one mirror deflects in theX-direction and the other deflects in the Y-direction. Tilting of themirrors is accomplished, for example, with the aid of galvanometercontrol elements. The fluorescent or reflected light coming from thesample passes in this mostly conventional descanning arrangement via thesame scanning mirror back to the beam splitter and passes the latter inorder subsequently to be focussed onto the detection stop, downstream ofwhich the detectors are located. Detection light which does notoriginate directly from the focussing region takes a different lightpath and does not pass the detection stop, and so point information isobtained which leads through scanning of the object to athree-dimensional image. Illumination and detection take place in thiscase on the objective side, that is to say on sides of the microscopeoptical system.

[0005] It is also possible in a transmitted-light arrangement for thefluorescent light or the transmitted light—the transmission of theexciting light-, for example, to be detected on the condenser side, thatis to say on the side of a condenser arranged downstream of the object.The detection-light beam then does not pass via the scanning mirrors tothe detector. Such an arrangement is denoted as a non-descanningarrangement.

[0006] In order to detect the fluorescent light, there would be a needin the transmitted-light arrangement for a condenser-side detection stopin order—as in the descanning arrangement described—to achieve athree-dimensional resolution. In the case of two-photon excitation,however, it is possible to dispense with a condenserside detection stop,since the probability of excitation is a function of the square of thephoton density or the intensity, which is naturally much higher at thefocus than in the neighbouring regions. The fluorescent light to bedetected therefore originates with high probability in overwhelmingproportion from the focussing region, and this renders superfluousfurther differentiation of fluorescence photons from the focussingregion from fluorescence photons from the neighbouring regions with theaid of a stop arrangement. However, the detection of the transmittedlight and/or the condenser-side detection of the fluorescent light canbe helpful in the case of single-photon excitation, as well.

[0007] Particularly against the background of a yield of fluorescencephotons which is low in any case for two-photon excitation, anon-descanning arrangement in which less light is generally lost on thedetection-light path is of interest.

[0008] It is known from EP 0 627 643 A1 to raise the signal yield byelectronic addition of the signals of the descanning and non-descanningdetectors. Two detection channels are thus superimposed electronicallyin this case.

[0009] In the known electronic addition or superimposition of thesignals, it is a problem that this form of electronic superimposition ofthe detector signals is complicated and slow. In particular, thedetectors used have to be set in a complicated fashion.

BRIEF SUMMARY OF THE INVENTION

[0010] It is therefore the object of the present invention to provide aconfocal scanning microscope which achieves a high signal yield and ahigh signal-to-noise ratio by the use of simple means.

[0011] The above object is achieved by a confocal microscope whichcomprises: a light source defining exciting light for illuminating anobject, a scanning device, a first and second detection light beinggenerated in the object wherein the detection light defines at least twodetection channels, a superimposing device for optically superimposingthe at least two detection channels and a detector assembly with atleast one detector for detecting the detection light.

[0012] In a way according to the invention, it has firstly been realizedthat raising the signal yield can be achieved not solely by electronicaddition of the signals of the detectors used. Also in a way accordingto the invention, optical superimposition of at least two detectionchannels by means of a superimposing device is provided for thispurpose. With the microscope according to the invention, it is possibleto dispense entirely with vulnerable electronic components which haveadditionally to be integrated in the known microscope. In this case, thecomplicated setting of the detectors used is also eliminated. Finally,the optical superimposition provides a quicker superimposition oraddition technique than the conventional electronic addition technique.

[0013] Consequently, the microscope according to the invention realizesa microscope in which a high signal yield and a high signal-to-noiseratio is achieved by simple means.

[0014] In concrete terms, the detection light of at least two detectionchannels could be detected in a common detector assembly having at leastone detector. However, it would also be possible to detect more than twodetection channels in a common detector assembly.

[0015] The location of the superimposition of the detection light of therespective detection channels is not prescribed in principle. In astructurally particularly simple design, it would be possible, however,for the detection light of the detection channels to be opticallysuperimposed in the detector assembly. In this case, the detection lightof the respective detection channels is fed to the detector assembly ina suitable way.

[0016] The detector assembly could be a descanning detector assembly, itbeing rendered possible in a practical way to make use for opticalsuperimposition of a descanning detector assembly frequently alreadypresent in a microscope. An additional detector assembly is thereforenot required.

[0017] The detector assembly could, however, also be a non-descanningdetector assembly. It would then be necessary when extending aconventional microscope which originally has a descanning detectorassembly to provide a further detector assembly with the aid of which itis then possible to detect the detection light of all the detectionchannels. In particular, it would be possible thereby for the detectionlight of at least one detection channel to be guided to the detectorassembly before it traverses—as originally planned—a scanning device.For this purpose, the detection light could preferably be guided to thedetector assembly by splitting between an objective and the scanningdevice—from the original beam path. For splitting purposes, a colourbeam splitter could, in a particularly simple way, be arranged in a beampath of the microscope.

[0018] In a concrete refinement, a first detection channel could exhibitdetection light which is emitted on the side of the object facing thelight source, and a second detection channel could exhibit detectionlight which is emitted on the side of the object averted from the lightsource. Expressed more accurately, the first detection channel couldexhibit reflected and/or fluorescent light. The second detection channelcould, by contrast, exhibit transmitted and/or fluorescent light.

[0019] The detector assembly could be assigned to the first detectionchannel. In other words, it could be a detector assembly which isalready present in the descanning arrangement in the case of aconventional microscope.

[0020] With regard to a concrete superimposition of two detectionchannels, the detection light of the second detection channel could beguided to the detector assembly by means of the superimposing device.This case could, in particular, concern an already present descanningdetector assembly.

[0021] In an alternative refinement, both the detection light of thefirst detection channel and the detection light of the second detectionchannel could be guided to the detector assembly by means of thesuperimposing device. In this case, the detector assembly could be adetector assembly which is provided specifically for the superimposingtechnique and which is, if appropriate, implemented in addition to adescanning detector assembly already present. The detector assemblycould be a nondescanning detector assembly, in particular.

[0022] With regard to effective optical superimposition of the detectionchannels, it could be possible to detect reflected light of the firstdetection channel and transmitted light of the second detection channeljointly, preferably in one and the same detector. Reflected light of thefirst detection channel and transmitted light of the second detectionchannel could in this case be approximately in the same wavelengthregion.

[0023] Furthermore, with reference to effective superimposition of thedetection channels, fluorescent light of the first detection channel andfluorescent light of the second detection channel could be detectedjointly, preferably in one and the same detector. Fluorescent light ofthe first detection channel and fluorescent light of the seconddetection channel could in this case be approximately in the samewavelength region.

[0024] Depending on the number of wavelength regions to be detected, itwould be possible to provide a plurality of detectors which could thendetect different wavelength regions.

[0025] The superimposing device could have a light-guiding device inorder to ensure reliable superimposition of the detection channels. In astructurally particularly simple way, the light-guiding device couldhave an optical fibre. Glass fibres, in particular, can be used in thiscase. In particular, the light-guiding device could have an opticalconductor filled with a liquid. The optical conductor filled with theliquid preferably has a large numerical aperture. Such light-guidingdevices are flexible and easy to handle. In concrete terms, alight-launching optical system and a light-output optical system couldbe assigned to the optical fibre or the optical conductor filled withthe liquid.

[0026] In an alternative refinement, the light-guiding device could haveat least one mirror. One or more lenses could be assigned to the mirroror the mirrors. This renders it possible to construct mirror/lensarrangements which likewise permit reliable light guidance.

[0027] With regard to a particularly selective detection of thedetection light, the detector assembly could be assigned alight-splitting device for splitting the detection light into differentwavelength regions. The light-splitting device could have at least onecolour beam splitter in a particularly simple way. In concrete terms, aplurality of colour beam splitters could be arranged in series in thiscase in order to render it possible to split different wavelengths orwavelength regions.

[0028] As an alternative or addition hereto, the light-splitting devicecould have at least one partially transparent mirror. A bandpass orblocking filter could be arranged downstream of this mirror or thesemirrors. In the case of the use of mirrors as splitting component, aswell, it would also be possible to arrange a plurality of such mirrorsin series, if appropriate with a downstream bandpass or blocking filter.Splitting the fluorescent light into a plurality of spectral regions isalso possible thereby.

[0029] As an alternative to the use of colour beam splitters or mirrors,it would be possible to use for splitting purposes a multiband detectorwhich is described, for example, in DE 199 02 625 A1. Splitting thefluorescent light into a plurality of spectral regions is also possiblewith the aid of such a multiband detector.

[0030] A laser could be used in a particularly advantageous way as lightsource. However, it is also conceivable to use other suitable lightsources.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0031] There are various possibilities of configuring and developing theteaching of the present invention in an advantageous way. For thispurpose, reference is made to the drawing. In the drawing:

[0032]FIG. 1 shows a diagrammatic illustration of a first exemplaryembodiment of a microscope according to the invention,

[0033]FIG. 2 shows a diagrammatic illustration of a second exemplaryembodiment of a microscope according to the invention, with a separatenon-descanning detector assembly, and

[0034]FIG. 3 shows a diagrammatic illustration of a third exemplaryembodiment of a microscope according to the invention, with a separatenon-descanning detector assembly.

DETAILED DESCRIPTION OF THE INVENTION

[0035]FIG. 1 shows a first exemplary embodiment of a microscopeaccording to the invention in a diagrammatic illustration. Themicroscope is a confocal laser scanning microscope. The microscope has alight source 1 designed as a laser. The light source 1 emits excitinglight 2 which is reflected to a scanning device 4 via a main beamsplitter 3. The scanning device 4 leads the exciting light beam througha microscope optical system or an objective 5 via an object 6.Transmitted light passing through the object 6 and fluorescent lightproduced in the object 6 pass through a condenser 7, forming acondenser-side detection light 8 in the process. Exciting light 2reflected by the object 6 and fluorescent light produced in the object 6form an objective-side detection light 9.

[0036] Furthermore, the condenser-side detection light 8 reaches via adeflecting mirror 10 a launching optical system 11 which launches thedetection light 8 into an optical fibre 12. Provided at the end of theoptical fibre 12 is an output optical system 13 from which there emergesoutput detection light 14 which is reflected to a colour beam splitter16 via a mirror 15. Also reflected onto the colour beam splitter 16 isthe objective-side detection light 9, which is guided via the scanningdevice 4. In other words, the detection light 9 passes via a descanninglight path.

[0037] The colour beam splitter 16 reflects the spectrally lower-waveregion of the light to a detector 19 via a focussing optical system 17.The spectrally higher-wave component of the detection light reaches adetector 20 via a focussing optical system 18. Use is made in this caseof a descanning detector assembly which has two detectors 19 and 20 andis already present in the case of conventional microscopes of the typementioned at the beginning.

[0038] In the exemplary embodiment illustrated in FIG. 1, thetransmitted light and the fluorescent light emitted at the rear of theobject are guided to the already present detectors 19 and 20 with theaid of an optical fibre 12 and detected there together with thereflected and fluorescent light running via the scanning device 4.

[0039] Use is made in this case of two detectors 19 and 20, light ofdifferent wavelengths being fed to the detectors 19 and 20 with the aidof a colour beam splitter 16. The wavelength regions of the transmittedlight guided to a detector or the fluorescent light emitted at the rearof the object preferably correspond in this case to the wavelengthregion of the reflected or fluorescent light to be detected with thisdetector.

[0040] In other words, a microscope is implemented which has a lightsource 1 which uses exciting light 2 to illuminate an object 6 to beinvestigated, at least two detection channels exhibiting detection light8 and 9 being produced. In this case, one detection channel exhibitsdetection light 8, and the other exhibits detection light 9. With regardto a high signal yield and a high signal-to-noise ratio, the microscopeis configured in such a way that at least two detection channels can beoptically superimposed by means of a superimposing device. In accordancewith FIG. 1, in concrete terms the superimposing device has a launchingoptical system 11, an optical fibre 12, an output optical system 13, amirror 15, a focussing optical system 17 and a focussing optical system18.

[0041] The spectrally lower-wave region of the fluorescent light of thedetection light 8 and the detection light 9 is optically superimposed inthe detector assembly. Moreover, the transmitted light and the reflectedlight are optically superimposed in the detector assembly.

[0042]FIG. 2 shows a diagrammatic side view of a second exemplaryembodiment of a microscope according to the invention. In essentialparts, the microscope corresponds to the first exemplary embodimentalready shown in FIG. 1. For this reason, identical components of thetwo exemplary embodiments are marked with the same reference numerals.

[0043] The second exemplary embodiment shown in FIG. 2 is a microscopewith an additional non-descanning detector assembly. Before it traversesthe scanning device 4, detection light 9 is guided in this case from afirst detection channel to the non-descanning detector assembly.Arranged for this purpose between the scanning device 4 and theobjective 5 is a colour beam splitter 21 which outputs the detectionlight 9 from the original beam path.

[0044] Provided for further guidance of the detection light 9 aremirrors 22 which lead the detection light 9 emitted on the objectiveside further to the colour beam splitter 16 into the detector assembly.The coupling of the detection light 8, which is emitted on the condenserside and exhibits transmission light and fluorescent light produced inthe object 6, is performed by analogy with the first exemplaryembodiment shown in FIG. 1. Instead of the detectors 19 and 20 used inthe first exemplary embodiment, detectors 23 and 24 are used in theexemplary embodiment shown in FIG. 2.

[0045] In the case of the optical superimposition of the detectionchannels, a combination is implemented which makes use, on the one hand,of an optical fibre 12 and, on the other hand, of a mirror arrangementwith mirrors 22.

[0046]FIG. 3 shows a diagrammatic illustration of a third exemplaryembodiment of a microscope according to the invention. The thirdexemplary embodiment shown in FIG. 3 is designed by analogy with thesecond exemplary embodiment shown in FIG. 2. The single differenceconsists in that the detection light 9 emitted on the objective side isguided to the non-descanning detector assembly via a launching opticalsystem 25, an optical fibre 26 and an output optical system 27—insteadof via mirrors. Otherwise, the design of the third exemplary embodimentcorresponds to the design of the second exemplary embodiment.

[0047] The detectors 19 and 20 are not used both in the second exemplaryembodiment and in the third exemplary embodiment.

[0048] The launching and output optical systems 11, 13, 25 and 27usually have lenses.

[0049] In the first exemplary embodiment shown in FIG. 1, thesuperimposing device is formed by the launching optical system 11, theoptical fibre 12, the output optical system 13, the mirror 15, thefocussing optical system 17 and the focussing optical system 18. In thesecond exemplary embodiment shown in FIG. 2, these components arefurther joined by the colour beam splitter 21 and the mirrors 22. In thethird exemplary embodiment shown in FIG. 3, by comparison with thesecond exemplary embodiment shown in FIG. 2, in the case of thesuperimposing device the mirrors 22 are replaced by the launchingoptical system 25, the optical fibre 26 and the output optical system27.

What is claimed is:
 1. A confocal scanning microscope comprising anobjective (5), a light source (1) defining exciting light (2) forilluminating an object (6), a scanning device (4), a first and seconddetection light (8, 9) being generated in the object (6) wherein thedetection light (8, 9) defines at least two detection channels, asuperimposing device (11, 12, 13, 15, 17, 18, 21, 22, 25, 26, 27) foroptically superimposing the at least two detection channels and adetector assembly with at least one detector (19, 20; 23, 24) fordetecting the detection light (8, 9).
 2. Confocal scanning microscopeaccording to claim 1 , wherein the first and second detection light (8,9) of the detection channels is optically superimposed in the detectorassembly.
 3. Confocal scanning microscope according to claim 1 , whereinthe detector assembly is a descanning detector assembly.
 4. Confocalscanning microscope according to claim 1 , wherein the detector assemblyis a non-descanning detector assembly.
 5. Confocal scanning microscopeaccording to claim 1 , wherein a beam splitter (21) is provided betweenthe objective (5) and the scanning device (4) for guiding the firstdetection light (9) to the detector assembly.
 6. Confocal scanningmicroscope according to claim 1 , wherein the first detection light (9),defining a first detection channel, is emitted on the side of the object(6) facing the light source (1), and the second detection light (8),defining a first detection channel, is emitted on the side of the object(6) averted from the light source (1).
 7. Confocal scanning microscopeaccording to claim 6 , wherein the detector assembly is assigned to thefirst detection channel.
 8. Confocal scanning microscope according toclaim 6 , characterized in that the second detection light (8) of thesecond detection channel is guided to the detector assembly by means ofthe superimposing device (11, 12, 13, 15, 17,18).
 9. Confocal scanningmicroscope according to claim 6 , wherein both the detection light (9)of the first detection channel and the detection light (8) of the seconddetection channel are guided to the detector assembly by means of thesuperimposing device (11, 12, 13, 15, 17, 18, 21, 22, 25, 26, 27). 10.Confocal scanning microscope according to claim 1 , wherein a pluralityof detectors (19, 20, 23, 24) are provided.
 11. Confocal scanningmicroscope according to claims 1, wherein the superimposing device (11,13, 15, 17, 18, 21, 22, 25, 26, 27) has a light-guiding device. 12.Confocal scanning microscope according to claim 11 , wherein thelight-guiding device is an optical fibre (12, 26).
 13. Confocal scanningmicroscope according to claim 11 , wherein the light-guiding device isoptical conductor filled with a liquid.
 14. Confocal scanning microscopeaccording to claim 13 , wherein the optical conductor filled with theliquid has a large numerical aperture.
 15. Confocal scanning microscopeaccording to claim 11 , characterized in that a light-launching opticalsystem (11, 25) and a light-output optical system (13, 27) are assignedto the light-guiding device.
 16. Confocal scanning microscope accordingto claim 1 , wherein a light-splitting device for splitting the firstand second detection light (8, 9) into different wavelength regions isassigned to the detector assembly.
 17. Confocal scanning microscopeaccording to claim 16 , wherein the light-splitting device has at leastone colour beam splitter (16).
 18. Confocal scanning microscopeaccording to claim 17 , wherein the light-splitting device has at leastone partially transparent mirror.
 19. Confocal scanning microscopeaccording to claim 16 , wherein the light-splitting device has amultiband detector.
 20. Confocal scanning microscope according to claims1, wherein the light source (1) is a laser.